Allergens in wine: Protein fining agents identified

Ezine

Published: Dec 3, 2013

Author: Steve Down

Channels: Base Peak

Fining agents

The quality of a wine relies not only on the quality of the grape but also on the techniques employed by the winemaker. There are times when the wine needs a helping hand if it becomes too hazy or bitter, for example. These incidents should not be viewed with dread by the production team because there is a group of well-established agents that can get the wine back on track.

Known as fining agents, they are designed to remove unwanted characteristics that affect the final product. Copper sulphate will remove thiols or hydrogen sulphide which impart the smell of rotten eggs or onions. Carbon is added to absorb those compounds which provide a brown off-colour.

Another set of traditional fining agents are proteins derived from milk or egg white. They are added to remove excess tannins by forming complexes to prevent the production of haze or the generation of deposits or off-flavours. Any leftover proteins are removed by adding another fining agent like the mineral bentonite.

The problem with using these proteins, typically casein from milk or lysozyme and albumin from egg white, is that they can cause allergies to susceptible people when they are consumed. As a result, the EU has followed the example of New Zealand and Australia in legislating that any products derived from egg or milk that are used as fining agents in winemaking must be declared on the label.

In parallel, there must be procedures in place that can test wines to see how much casein, lysozyme and albumin remains in the final product. To this end, Italian researchers from Bari have developed a high-resolution mass spectrometric method that measures the characteristic peptides produced by the proteins when they are broken down by trypsin.

High resolving power differentiates peptides

Linda Monaci and colleagues from the Institute of Sciences of Food Production and the University of Bari "Aldo Moro" chose a novel approach which combines LC/MS with the use of isotopically labelled peptides of the proteins to estimate the yield of tryptic peptides produced from the wine-based proteins.

Initially, they identified suitable marker peptides in the tryptic digests of ovalbumin, egg white powder or a commercial fining agent based on egg white. The mass spectrometer was operated in full-scan mode at high resolution to find candidate peptides, followed by a second scan in which all of the ions were transferred to a cell for high-energy collisional dissociation. The product ions were associated with the relevant precursor ion using the retention times and the extracted ion chromatograms. In this way, three characteristic doubly charged ions were selected for ovalbumin, two for lysozyme and three for casein, with two from αS1-casein and one from β-casein.

Then three different white wines made from Falanghina, Pinot grigio and Greco di Tufo grapes were fortified with the caseinate or egg white powder. Aliquots of the wines were subjected to ultrafiltration to extract the proteins and any potential low-molecular-weight interferences were removed by dialysis. Finally, following reduction and alkylation of the extracted proteins, the proteins were digested with trypsin to produce the marker peptides.

Allergens measured in wine

The efficiency of the wine ultrafiltration step was estimated using 15N-labelled analogues of two peptides, one derived from αS1-casein and the other from ovalbumin. The values were 20 and 36% for the two proteins, respectively. The same labelled peptides were employed to check the digestion efficiency with trypsin, which were estimated at 55 and 100% for αS1-casein and ovalbumin, respectively.

The overall method efficiencies calculated by combining these two factors or by spiking the final digest with a labelled peptide just before LC/MS analysis were both about 20%.

Based on the responses of the calibration graphs for each peptide, it was also apparent that one specific peptide could be monitored for all of the target proteins, whether they originated from egg white or caseinate. This would save having to check for more than one protein in the first instance, speeding up the whole testing process.

The ability of high-resolution mass spectrometry to identify specific peptides accurately rules out the possibility of interferences from other compounds of similar m/z values and instils confidence in the procedure. The detection limits of the proteins, based on the peptide markers, were 0.4-1.1 µg/mL. These are around the levels at which they can induce an allergenic response after drinking a 200 mL glass of wine, so the method has the correct sensitivity for practical applications.